Project Summary This MIRA proposal aims to solve critical gaps in knowledge of two poorly understood protein systems that are linked to health and human disease. To accomplish this proposal, the designed studies combine structural, inorganic, and biochemical approaches with an innovative metallocentric point of view that is essential yet has remained chiefly unexplored for these proteins. The first proposed system of study is the ferrous (Fe2+) iron uptake (Feo) system, which is present in nearly all bacteria and is used by pathogens to establish infection in mammalian hosts. Previous studies on Feo have either been too large or too small in scope, leading to a fragmented and inconclusive understanding with little insight into mechanism. This proposal outlines a comprehensive approach to study the Feo system at the protein level. Leveraging structural, spectroscopic, and biochemical analyses, this proposal aims to delineate the mechanism of prokaryotic Fe2+ transport, which will position future researchers to explore the urgent but broadly impactful possibility that Feo may be exploited to combat bacterial virulence. The second proposed system of study focuses on the arginine transferases (known as ATE1s), which are enzymes that arginylate the N-terminus of peptides or proteins, subsequently triggering their degradation via the ubiquitin-proteasome system. Normal ATE1 function is critical for neurogenesis and cardiovascular development, but structural and mechanistic details of ATE1-mediated arginylation are sorely lacking, prohibiting the targeting of this system for therapeutic intervention. Exciting results indicate ATE1s may be iron-containing enzymes, but the function of iron in this system remains unknown. This proposal aims to delineate the structure and mechanism of ATE1s, including the potential regulatory role of iron in these enzymes. To achieve this goal, this proposal combines protein- level structural, biochemical, and spectroscopic methods to elucidate the arginylation mechanism of ATE1s, and to resolve how iron controls this process. Once determined, this molecular-level detail will be invaluable to design small molecules that target ATE1 for intervention. Combined, the results from this proposal hold the promise to aid in the development of therapeutics to abrogate bacterial virulence and to treat neurological and cardiovascular diseases.